C677T and A1298C Mutations in the MTHFR Gene and Survival in

C677T and A1298C Mutations in the MTHFR Gene and
Survival in Colorectal Cancer
Gelu Osian1, Lucia Procopciuc2, Liviu Vlad1, Cornel Iancu1, Teodora Mocan1, Lucian Mocan1
1) 3rd Surgical Department; 2) Biochemistry Department, University of Medicine and Pharmacy “Iuliu Hatieganu” ClujNapoca, Romania
Abstract
Background and aims: Our preliminary results
laboratory have shown some association between C677T and
A1298C MTHFR mutations and factors influencing survival
in colorectal cancer. We studied the survival of patients
with colorectal cancer depending on the initial DukesMAC stage of the disease at the time of diagnosis and the
MTHFR mutation present. Methods: We randomly selected
69 patients with sporadic colorectal cancer who underwent
surgery at the Surgical Clinic III Cluj between October
2003 and May 2005. The study ended on 15 March 2008.
Survival data was verified in 48 cases. Survival analyses
were performed using Kaplan-Mayer survival curves and
median survival time was calculated. The comparison of
two or more categories was performed using the Logrank
test, considering the threshold value p≤0.05. Results: In
both stage B and C patients with the CT/TT mutation have
a poorer survival rate than those with the wild CC genotype
(p<0.05). The presence of the C677T mutation (CT or TT
genotype) in patients diagnosed in stage D did not result as
a significant survival risk factor (HR=0.537, 95% CI 0.1282.184) p>0.05. Patients diagnosed with stage C colorectal
cancer, who have the 1298C allele, have significantly better
survival than those without this allele, 60% vs. 15.4%,
(p=0.0016). Conclusions: In our study in both stage B and
C, patients with the CT/TT mutation have poorer survival
than the wild CC genotype. In stage B patients, the A1298C
mutation is a negative prognostic factor. The presence of the
A1298C mutation in a hetero- or homozygous form plays a
protective role in stage C.
Key words
MTHFR – colorectal cancer – survival.
Received: 24.06.2009Accepted: 10.11.2009
J Gastrointestin Liver Dis
December 2009 Vol.18 No 4, 455-460
Address for correspondence:
Gelu Osian
3rd Surgical Department
19-21 Croitorilor Str.
400162 Cluj-Napoca, Romania
E-mail: geluosian@yahoo.com
Introduction
The DNA replication error is one of the main mechanisms
involved in colorectal carcinogenesis. Proteins that stop
DNA synthesis and remove defective sequences play a
role in stopping the error transmission. These proteins
are the product of MMR (mismatch repair) genes [1].
DNA methylation plays an important role in the repair of
replication errors and the regulation of gene expression.
Thus, by methylation, DNA is resistant to the action of
endonucleases, a control for the maintenance of healthy
DNA is obtained.
DNA methylation requires the addition of a methyl
group, whose donor is S-adenosyl methionine [2-4]. Its
formation depends on the dietary folate intake and on the
activity of the enzyme 5,10 methylenetetrahydrofolate
reductase (MTHFR). This catalyzes the reduction of 5,10
methylenetetrahydrofolate to 5 methyltetrahydrofolate,
which participates in the remethylation of homocysteine
to methionine, from which S-adenosyl methionine will be
synthesized.
The gene encoding MTHFR is situated on the short arm
of chromosome 1 (1p36.3). Of the known MTHFR mutations
detected in this gene, two have an effect on the enzymatic
activity: the C677T variant, in which the nucleotide at
position 677 is modified and leads to the replacement of
alanine by valine at that enzyme position, and the A1298C
variant, in which the nucleotide at position 1298 is modified
and leads to the replacement of glutamate by alanine at that
position. The (homozygous) 677TT genotype leads to a
decrease in MTHFR activity [5], which causes a decrease in
DNA repair in the excision areas of the uracyl group [6, 7].
The A1298CC variant has no major effects on the enzyme
activity [8].
Individuals with a homozygous 677TT genotype have
an activity of the MTHFR enzyme of 30% compared to the
wild variant [9], 677CT heterozygotes have a 65% activity
of the enzyme [9]. Individuals homozygous for the 1298CC
mutation have a 40% activity [8].
The studies of the MTHFR genotype in patients with
colorectal cancer have mainly evaluated the C677T
456
mutation. The majority report a protective effect of the
C677T mutation, with an OR between 0.54-0.80 [10-13],
but there are also studies that have evidenced an increase
in the risk of colorectal cancer in these patients with an OR
between 1.09-1.44 [14-16]. Studies of the A1298C mutation
also note its protective effect, with a reduction in the risk
of appearance of colorectal cancer for CC homozygotes
compared to AA homozygotes, with OR values ranging
between 0.6 and 0.8 [17-20].
The folates play the central part in the metabolism of
the methyl group, being an important coenzyme for both
the DNA methylation and its synthesis.
Genetic studies support the idea that low body folate
levels are predisposing to various neoplasms [21], the most
frequently reported being colorectal cancer [22]. Moreover,
a high supply of folates seems to have a protective effect
against cancer. The relationship between the MTHFR
mutations and diet is considered to be an example of
interaction between environmental and genetic factors in
the colorectal tumor genesis [23-25].
Folate deficit induces chromosome ruptures, a condition
associated with an increased cancer risk [21]. The molecular
mechanism consists of the enhanced incorporation of uracyl
into the DNA, the repair being followed by the break of
a DNA chain, and if there are two adjacent uracyl bases,
ruptures of both chains may occur [21, 26]. This mechanism
was confirmed by in vitro studies of human colon epithelial
cells [27].
One of the first studies regarding the protective effect
of the C677T mutation, which evidenced the decreased
risk of colorectal cancer in homozygotes (TT) depending
on the exposure to environmental factors, was published
by Ma et al in 1997 [10]. The protective effect of the TT
mutation is achieved by the increase of the cell content of
5,10-methyl THF that will facilitate the nucleotide synthesis.
The protective effect occurs with sufficient folate supply.
Subsequent studies have evidenced increased colorectal
cancer risk in homozygotes with low folate supply [28].
Contrary to these reports our previously published
studies showed an increase in the risk of colorectal cancer
in patients homozygous for the two mutations with OR=2.13
for the C677T mutation, OR=3 for the A1298C mutation,
respectively [29]. At the same time, we found a statistically
significant correlation of the A1298C mutation with a
number of factors with a prognostic role in colorectal cancer:
lymph node invasion, pT stage, Dukes-MAC stage, results
which suggested a possible prognostic value of MTHFR
mutations in colorectal cancer.
Working hypothesis
Starting from preliminary results that evidenced the
association of mutations with prognostic factors, we may
formulate the hypothesis that these mutations influence
individually, as prognostic factors, survival in colorectal
cancer. We decided to study the survival of patients with
colorectal cancer depending on the initial Dukes-MAC
stage of the disease at the time of diagnosis and the MTHFR
mutation present. These results will show the concrete value,
Osian et al
as prognostic value, of the gene variants in the studied
group.
Material and method
Patient selection
We randomly selected 69 patients with sporadic
colorectal cancer operated at the Surgical Clinic III Cluj
between October 2003 and May 2005. The selection criteria
were the histological confirmation of cancer and surgery
in the Surgical Clinic III Cluj. The exclusion criteria were
a personal or family history of familial adenomatous
polyposis or hereditary non-polyposis cancer and a history
of inflammatory bowel disease. We closed the study on 15
March 2008. We obtained data on survival only in 48 cases,
which are analyzed in what follows.
The colonoscopies were performed in the Medical
Clinic III Cluj – Department of Digestive Endoscopy.
The histopathological examinations were carried out in
the Department of Pathological Anatomy of the “Prof. Dr.
Octavian Fodor” Clinical Hospital Cluj-Napoca.
Identification of MTHFR mutations
Peripheral blood samples were taken and DNA was
extracted from leukocytes using Lahiri’s method (30). The
C677T polymorphism located in exon 4 of the MTHFR gene
was examined by DNA amplification by PCR (the sense primer
had the sequence: 5’- ACCCACAGAAAATGATGCCCAG3’; the antisense primer had the sequence: 5’TGCCCCATTATTTAGCCAGGAG-3’) (Sigma Genosys),
and restriction fragment length polymorphism (PCRRFLP technique). The same technique was used for the
identification of the A1298C polymorphism located in exon 7
of the MTHFR gene (the sense primer had the sequence: 5’CACTTTGTGACCATTCCGGTTT-3’; the antisense primer
had the sequence: 5’- CTTTGGGGAGCTGAAGGACTA3’) (Sigma Genosys).
C677T polymorphism was identified by the enzymatic
digestion of the fragment obtained by PCR amplification
(198pb) with the HinfI restriction endonuclease (New
England Biolabs). The C677T polymorphism creates a
restriction site for the Hinfl enzyme. The enzymatic digestion
was obtained in 10µl reaction mixture and included the
following reagents: 1X NE buffer (50 mM NaCl, 10 mM
Tris-HCl, 10 mM MgCl2, 1 mM DTT, pH 7.9@ 25ºC), and
5 units HinfI/ 10µl reaction mixture. Enzymatic digestion
was performed by incubating the mixture at 370C for 3
hours. The products of enzymatic digestion were separated
by electrophoresis in 3% agarose gel stained with ethidium
bromide. By enzymatic digestion, the normal allele with
cytosine in position 677 (C677) forms a fragment undigested
by 198pb, while the mutant allele with thymine in position
677 (T677) form two fragments of 175 and 23pb.
A1298C polymorphism was identified by enzymatic
digestion of the fragment obtained by PCR amplification
with Mboll restriction endonuclease (New England Biolabs).
A1298C polymorphism annuls a restriction site for the Mboll
MTHFR mutations and survival in colorectal cancer
enzyme. Enzymatic digestion was obtained in 10µl reaction
mixture and included the following reagents: 1X NE buffer
(50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 1 mM
DTT, pH 7.9@ 25ºC), and 5 units MboII/ 10µl reaction
mixture. Enzymatic digestion was performed by incubating
the mixture at 370C for 3 hours. The products were separated
by electrophoresis in 3% agarose gel stained with ethidium
bromide. The normal allele with adenine in position 1298
(A1298) forms by enzymatic digestion fragments of 84, 31,
30, 28pb, while the mutant allele with cytosine in position
1298 (C1298) forms fragments of 56, 31, 30 and 28pb.
We use these methods also used in our previously
published study [29].
Collection of data
Data were collected prospectively and retrospectively by
specially trained resident doctors, who obtained informed
consent of the studied patients.
Data on survival were obtained by the periodic
monitoring of patients through the outpatient service of the
Surgical Clinic III Cluj, deaths being confirmed over the
phone by the patients’ families and verified with the official
registry of the National Population Evidence Service.
Statistical analysis
We compared the survival of patients with the wild gene
variant to the group of patients who presented the mutation
in a hetero- or homozygous form.
Analyses were performed using the actuarial method
(Kaplan-Mayer survival curves) and including the calculation
of the median survival time. The comparison between two or
more categories was done using the Logrank test, considering
the threshold value p≤0.05. In the case of the comparison of
two groups of patients, the analysis was completed with a
proportional hazards model with HR (CI 95% ) calculation.
Standard HR interpetation, as an estimated relative risk of
the event of interest occurring in one group compared to the
other group was used. (HR(CI95%)>1-risk effect, HR (CI
95%) <1-protective effect).
457
Table I. General characteristics of the patients in Dukes-MAC
stages B, C and D (48 cases)
Characteristics
Total
(48 cases)
Gender
Female (%)
23
(47.91%)
Male (%)
25
(52.09%)
Age (years)
± SD
65.86 ±
9.62
Median
64.9
DukesMAC B
(19 cases)
DukesMAC C
(18 case)s
DukesMAC D
(11 cases)
-
-
-
-
-
-
C677T
genotype
CC
25
11
9
5
CT or TT
23
8
9
6
A1298C
genotype
AA
22
7
13
2
AC or CC
26
12
5
9
Stages B and C
The survival curves of patients with the MTHFR C677T
mutation diagnosed in stages B and C are shown in Fig. 1.
Results
The general characteristics of the studied patients are
presented in Table I. The genotype distribution was in the
Hardy-Weinberg equilibrium.
In relation to Dukes-MAC stages, mortality in the studied
interval was as follows: A=0%, B=31.58%, C=68.42% and
D=81.81%. Because the mortality in the Dukes-MAC stage
A was 0%, in order to avoid any confusion we do not present
or discuss any data about patients from this group.
We obtained data regarding survival until the study was
closed in 48 cases: 19 cases diagnosed in stage Dukes-Mac
B, 18 cases in stage C and 11 cases in stage D.
Our results showed that cancer mortality of the studied
group, regardless of stage, was not linked to MTHFR
genotypes.
We analyzed survival separately for each stage and each
mutation.
Fig 1. Comparative survival curves between Dukes-MAC B and
C stages depending on the C677T genotype.
In stages B and C, patients with the CT/TT mutation
had poorer survival than those with the wild CC genotype.
(p=0.0221) (Table II).
For the A1298C genotype (Fig. 2) we found that survival
in stage B was similar to the C677T mutation. The presence
of the A1298C mutation in a hetero- or homozygous form
played a protective role for patients in stage C (p=0.0016)
(Table II).
458
Osian et al
Table II. Survival related to Dukes-MAC stage and MTHFR genotype
Stage
Dukes B
Dukes C
Dukes B
Dukes C
Dukes D
Dukes D
Genotype
Survival at
6 months*
Survival at
12 months*
Survival at
24 months*
Survival at
48 months*
HR
95% CI
P
Chi
Square
677CC
1
0.900
(0.095)
0.800
(0.126)
0.700
(0.145)
2.782
0.513
0.427
677CC/TT
0.875
(0.117)
0.750
(0.153)
0.625
(0.171)
0.625
(0.171)
0.783
5.637
677CC
0.667
(0.157)
0.444
(0.166)
0.333
(0.157)
0.333
(0.157)
3.210
0.0221
9.6173
677CC/TT
0.444
(0.166)
0.333
(0.157)
0.222
(0.139)
0.222
(0.139)
1.245
6.823
1298AA
0.833
(0.152)
0.833
(0.152)
0.833
(0.152)
0.833
(0.152)
0.411
0.401
0.704
1298AC/CC
0.917
(0.080)
0.833
(0.108)
0.667
(0.136)
0.583
(0.142)
0.087
2.654
1298AA
0.538
(0.138)
0.231
(0.117)
0.154
(0.100)
0.154
(0.100)
0.411
0.0016
15.2319
1298AC/CC
1
0.800
(0.179)
0.600
(0.219)
0.600
(0.219)
0.236
0.623
677CC
0.800
(0.179)
0.600
(0.219)
0.400
(0.219)
0.400
(0.219)
0.5371
0.3671
0.8135
677CT/TT
0.833
(0.152)
0.667
(0.192)
0.333
(0.192)
0.167
(0.152)
0.1208
2.1846
1298AA
0.500
(0.354)
0.500
(0.354)
0.500
(0.354)
0.500
(0.354)
0.6461
0.6699
0.1817
1298AC/CC
0.778
(0.139)
0.556
(0.166)
0.222
(0.139)
0.222
(0.139)
0.1035
–
4.2960
* Values were expressed as survival proportion (standard error)
impact for patients diagnosed in stage D, being associated
with poorer survival (Table II).
The analysis of the A1298C mutation evidences a
peculiarity: patients with the mutation present (AC/CC)
Fig 2. Comparative survival curves between Dukes-MAC B and
C stages depending on the A1298C genotype.
Stage D
Figure 3 shows the survival curves for the C677T
mutation.
The presence of the C677T mutation had a negative
Fig 3. Comparative survival curves between DukesMAC D stage depending on the C677T genotype.
MTHFR mutations and survival in colorectal cancer
initially have better survival, as a percentage of the group,
until 3 months postoperatively, but this subsequently
decreases and patients with a wild genotype finally have a
better survival (Fig. 4, Table II).
Fig 4. Comparative survival curves between DukesMAC D stage depending on the A1298C genotype.
Discussion
The analysis of the correlation of the MTHFR genotype
with mortality and survival represents the main element
of evaluation of their prognostic significance in colorectal
cancer.
Regarding overall mortality, we found no statistically
significant correlations. Patients with the A1298C mutation
have better survival than patients with a wild genotype, the
risk of death being OR=0.721, but statistical significance
cannot be confirmed.
In both stage B and C, patients with the CT/TT mutation
present have poorer survival than those with the wild CC
genotype (p<0.05). The presence of the C677T mutation
has a negative impact in patients diagnosed in stage D, its
presence being associated with poorer survival-17.7 % vs.
40% at 25 months but this was not statistically significant
(HR=0.5371, 95%IC 0.1208- 2.1846, p>0.05).
In stage B, the survival of patients with the A1298C
mutation (AC/CC) is similar to the C677T mutation, so
patients diagnosed in this stage have poorer survival;
consequently the mutation is a negative prognostic factor for
this group. Patients diagnosed with stage C colorectal cancer,
who present the 1298C allele, have better survival than those
without this allele, 60% vs. 15.4% at 27 months, statistically
significant p=0.0016. So, the A1298C mutation may be a
positive prognostic factor in stage C. In stage D, during
459
the first 3 months, the A1298C mutation brings a survival
benefit (77% compared to 50%); subsequently the patients
of this group have a much lower survival rate compared to
those with the wild genotype. Overall, the influence of the
A1298C mutation is unfavorable in stage D.
A number of recent studies have evaluated the correlation
of the MTHFR polymorphism with survival in colorectal
cancer. Derwinger et al [31], by studying the C677T
mutation in 544 cases of colorectal cancer, reported that its
presence may affect sensitivity to 5-FU chemotherapy and
the risk of side effects, so that survival in stage III, possibly
in stage IV is affected. In as much as we can associate stage
Dukes-MAC C with stage TNM III and stage D with stage IV,
our results appear similar. However, it should be mentioned
we evidenced no relationship between the sensitivity to 5FU chemotherapy and MTHFR polymorphism. Capitain et
al [32], by evaluating 76 cases of colorectal cancer, shows
an increase in side effects and a shorter survival time in
patients with the A1298C mutation. Yoshiya et al [33] found
no correlations between the C677T mutation and survival
in 114 cases of colorectal cancer.
We are, however, aware of our study limits, especially
regarding the number of cases. Chosen sample size can
induce testing power decrease for part of the analyses. For
this reason analyses were not further adjusted to prevent
even more restrictions of sample size per subgroups. Further
research is needed, with extended sample size, to validate/
invalidate present results.
Conclusions
We report that MTHFR C677T and A1298C mutations
are significantly associated with survival in our small
study.
For patients diagnosed with Dukes stage B and C, the
CT/TT mutations are associated with a significantly poorer
survival than those with the wild CC genotype. The presence
of this mutation may be a negative prognostic factor.
In stage B patients, the A1298C mutation may be a
negative prognostic factor. The presence of the A1298C
mutation in a hetero- or homozygous form plays a protective
role in stage C patients.
Overall, the influence of the A1298C mutation is
unfavorable in stage D.
Conflicts of interest
Nothing to declare.
Acknowledgement
This study was financed through a VIASAN grant
(VIASAN 363), project manager Dr. Lucia Procopciuc.
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